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Immune cell profiles in peripheral blood have been associated with bladder cancer outcomes, however, their association with response to immunotherapy and the tumor microenvironment is a major unresolved issue. Although tumor growth can be attenuated via the activation of tumor-infiltrating effector T cells, the relationship between tumor infiltration and immune activation remains unclear. This study explored the interaction between bladder cancer outcomes and immune profiles within peripheral blood and a tumor microenvironment (TME) based on DNA methylation profiles. Peripheral blood and the matched tumor FFPE DNA methylation profiles of 60 non-muscle-invasive bladder cancer (NMIBC) and 12 muscle-invasive bladder cancer (MIBC) patients. Cell-type deconvolution approaches were applied to estimate 12 peripheral immune cell-type proportions and 17 cell-type proportions within TME. We found a positive correlation between dendritic cell proportions in the TME with peripheral CD8T memory cell proportions (r = 0.35, P = 0.003) and a negative correlation between dendritic cell proportions in the TME with peripheral regulatory T cell proportions (r = -0.28, P = 0.021). In addition, monocyte cell proportions in TME had a positive correlation with peripheral B memory (r = 0.37, P = 0.002) and CD8T memory cell proportions (r = 0.43, P = 0.0002). To investigate associations of bladder cancer outcomes with immune cell profiles, using Cox proportional hazard models, we observed an association between the fraction of dendritic cells and the hazard of death (HR = 1.27, 95% CI = 1.06-1.53). Further, a high endothelial cell proportion was significantly associated with an increased hazard of death and tumor recurrence (HR = 1.06, 95% CI = 1.01-1.13) in TME. In addition, the peripheral neutrophil-to-lymphocyte ratio (HR = 1.49, 95% CI = 1.01-2.22), monocyte (HR = 1.17, 95% CI = 1.05-1.31), neutrophil (HR = 1.04, 95% CI = 1.01-1.07), and basophil (HR = 1.35, 95% CI = 1.01-1.81) cell proportions were associated with an increased hazard of death and tumor recurrence. Our results integrated the information on bladder cancer outcomes and cell profiles in TME and peripheral blood, providing biomarkers for estimating bladder cancer prognosis using genome-scale DNA methylation measures. Citation Format: Ji-Qing Chen, Lucas A. Salas, John K. Wiencke, Devin C. Koestler, Annette M. Molinaro, Angeline S. Andrew, John D. Seigne, Margaret R. Karagas, Karl T. Kelsey, Brock C. Christensen. Integration of associations of immune profiles in peripheral blood and tumor microenvironment with bladder cancer outcomes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6675.

Background: Genome-wide association studies (GWAS) revealed that 25 regions in 24 genes are associated with adult diffuse glioma development. These regions were identified by performing GWAS of glioma overall and by pathology (GBM and nonGBM). The 2016 WHO Classification of Tumors of the Central Nervous System utilizes two somatic alterations to molecularly-classify adult diffuse glioma: IDH mutation and 1p/19q codeletion. We hypothesized that germline variants may increase susceptibility to, or interact with, these somatic alterations to accelerate the development of specific molecular subtypes of glioma. We further hypothesize that germline variants associated with IDH-mutated glioma might be associated with other IDH-mutated tumors, namely, cholangiocarcinoma, acute myeloid leukemia (AML) and melanoma. Methods: We performed a GWAS by glioma molecular subtype - as defined by presence or absence of IDH somatic mutation and 1p/19q codeletion. A total of 1320 glioma cases and 1889 controls were used in the discovery set, and 799 glioma cases and 808 controls in the validation set. A meta-analysis was performed with a genome-wide p-value threshold of 5 × 10−8. GTEx data were used to perform an expression quantitative trait loci (eQTL) analysis. For germline variants that were significantly associated with IDH-mutated glioma, we evaluated pleiotropy with cholangiocarcinoma, AML and melanoma using TCGA and Mayo Biobank controls. Results: Variants in or near D2HGDH on chromosome 2 were genome-wide significant in IDH-mutated glioma (meta p-value = 2.82 × 10−10). TCGA reported that the D2HGDH region was commonly deleted in IDH-mutated gliomas that do not have 1p/19q codeletion. In TCGA data for IDH-mutated, non-codeleted glioma, we observed that the D2HGDH variant was inversely associated with tumor deletions of D2HGDH (odds ratio=0.57, p-value=0.015). The eQTL analyses demonstrated significant associations between D2HGDH germline variant and expression of D2HGDH (p=2.2 × 10−11). Further stratifying IDH-mutated glioma by 1p/19q codeletion status, one variant near FAM20C on chromosome 7 was genome-wide significant in gliomas that have IDH mutation and 1p/19q codeletion (meta p-value=9.56 × 10−9). Analyses are currently underway to evaluate pleiotropy of these IDH-mutated glioma germline variants with other IDH-mutated tumors including cholangiocarcinoma, AML and melanoma. Variants in or near GMEB2 on chromosome 20 were genome-wide significant in IDH wild-type glioma (meta p-value=2.60 × 10−10). The most significant variant in the GMEB2 region remained significant after adjustment for the known RTEL1 glioma risk variant nearby on chromosome 20 (p=0.029). Conclusions: We identified and validated novel germline variants in two genes that are associated with etiology of IDH-mutated and one gene that is associated with IDH wild-type adult diffuse glioma. Citation Format: Jeanette E. Eckel-Passow, Kristen L. Drucker, Thomas M. Kollmeyer, Matthew L. Kosel, Paul A. Decker, Annette M. Molinaro, Terri Rice, Corrine E. Praska, Lauren E. Clark, Alissa A. Caron, Alexej Abyzov, Anthony Batzler, Jun S. Song, Melike Pekmezci, Helen M. Hansen, Lucie S. McCoy, Paige M. Bracci, Joseph Wiemels, John K. Wiencke, Stephen Francis, Terence C. Burns, Caterina Giannini, Daniel H. Lachance, Margaret Wrensch, Robert B. Jenkins. Adult diffuse glioma GWAS by molecular subtype identifies variants in D2HGDH, FAM20C and GMEB2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1193.

Glioma patients demonstrate abnormalities in peripheral blood leukocytes that have been associated with survival time. Here we assessed immune cell profiles in archival blood samples obtained 5-25 years ago using a novel epigenetic approach called immunomethylomics. We first validated the approach to measure the proportions of CD4 T, CD8 T cells, B cells, NK cells, neutrophils, and monocytes in archival blood using the new 850,000 feature EPIC Illumina methylation bead array. The immunomethylomic assay was shown to match the performance of multiparametric flow cytometry and thus is a highly accurate method for immune profiling. We next measured cell profiles in blood from 312 molecularly defined AGS subjects. In this cohort we enriched for patients with triple negative tumor subtypes (IDH wildtype, 1p19q negative, TERT non-mutant). Elevations in the neutrophil lymphocyte ratio (NLR) significantly increased with grade, whereas the proportions of T and B cells decreased with increasing grade. Using an established cut point of > 4.0 for the NLR revealed that this immune parameter was associated with shorter survival times in glioblastoma (median overall survival (mOS) 12.6 vs. 16.9 months) and non-glioblastoma (mOS 16.7 vs. 36 months) patients. Ongoing analyses of the cohort will be presented to evaluate the effects of age, gender, surgery, chemoradiation and tumor grade on the survival results. Because DNA based immune cell profiles do not require intact cells nor preserved proteins, they provide a powerful tool to glean potentially important immunologic information from historic stored samples that would otherwise be useless using current cytometry-based approaches. Citation Format: John K. Wiencke, Annette Molinaro, Warrier Gayathri, Jennifer Clarke, Jennie Taylor, Devin Koestler, Joe Wiemels, Helen Hanson, Lee Sean, Terri Rice, Lucie McCoy, Lucas Salas, Wrensch Margaret, Brock Christensen, Karl T. Kelsey. Immune profiles in the San Francisco Adult Glioma Study using Immunomethylomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5052.

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic. Cancer Res; 72(7); 1773–83. ©2012 AACR.

Background: Recently it has been shown that biospecimen handling and pre-analytical variables can dramatically affect biomarker assays of protein expression in tumor tissue. Phospho-proteins and even labile unmodified proteins have been suggested to show significant loss of expression due to prolonged time to formalin fixation. Here we assess 4 clinically relevant proteins (ER, PR, HER2 and Ki67) and 20 other proteins for changes as a function to the key preanalytic variables of ischemic time. The ultimate goal of our effort is to find a method to monitor the degradative effect of these variables by construction of a Tissue Quality Index (TQI). Materials and Methods: Two different breast cancer cohorts were used in order to analyze the biomarkers and their change according to time to formalin fixation. The first cohort consists of 93 breast cancer specimens in 2 fold redundancy on a TMA with cell lines and controls. The time to formalin fixation for each breast cancer specimen was recorded and ranges from 25 to 415 minutes. The second cohort consists of 25 matched pairs of breast cancer biopsies and resections. The time to formalin fixation for the biopsies is minimal while the time to fixation for the resections, though not recorded, averages between 1 and 3 hours. Protein expression was measured using the AQUA method of quantitative immunofluorescence. Results: ER alpha, PR, HER2 and Ki67 were each analyzed on the time to fixation array with 2 different antibodies commonly used in the clinical setting. Correlation of AQUA scores of these markers with time to formalin fixation revealed a trend towards loss of protein expression as a linear function of time to fixation without reaching statistical significance. Analysis of these 4 proteins on the matched pairs of biopsies and resections showed that tumor heterogeneity predominated over the effects of ischemic time. Toward identification of markers for a TQI, 20 biomarkers were analyzed on the time to fixation array. Both HIF1alpha and AKAP13 show a significant increase as a function of time to fixation, whereas pMAPK, histone 4 and pTyrosine 4G10 revealed a significant loss of expression. These trends were confirmed in the matched pair validation set with the execption of histone 4. A TQI is being built from these variables. Conclusions: Ischemic time is a critical pre-analytical variable that impacts measurement of protein expression in tumor tissue. The 4 standard markers used clinically in breast cancer appear to show only moderate effects that appear less critical to measurement accuracy than the issue of tumor heterogeneity. We identified 4 proteins which show a significant change with increasing time to formalin fixation and should allow construction of a TQI for assessment of pre-analytic antigenic degradation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-03.

Purpose. Activation of the PI3K/mTOR pathway in low-grade gliomas (LGG) provides a rationale for treatment with mTOR inhibitors as in preclinical studies activation of PI3K/AKT/mTOR renders cells more susceptible to mTOR inhibition. In a phase II prospective clinical trial of the mTOR inhibitor everolimus for recurrent LGGs, we performed preliminary analyses to identify early imaging and molecular biomarkers of treatment response. Methods. Thirty-six patients (of a planned 60 patients) underwent biopsy at the time of recurrence and were then treated with everolimus. Serial multimodal magnetic resonance imagining was done every 2 months; the imaging included anatomic, dynamic contrast enhanced (DCE), perfusion-weighted, diffusion-weighted, and MR spectroscopic imaging. Tumor molecular analyses included IDH1R132H mutations, 1p19q co-deletions, and phosphoprotein analyses of activation of the PI3K/AKT/mTOR and MAPK pathways by immunohistochemistry, including p-S6, p-PRAS40, p-4EBP1, and p-MAPK. Results. Histologies at original diagnosis were oligodendroglioma (14), astrocyomta (9), oligoastrocytoma (13). At time of recurrence 9/36 patients had transformed to higher-grade disease. Therapy prior to study participation included radiation in 11 and temozolomide in 23. Twelve patients continue on active therapy, 15 have progressed, and the remaining discontinued drug due to toxicity (n=2), poor follow-up (n=1), refusal of further therapy (n=4), or unrelated intercurrent disease (n=2). Fifteen patients had stable disease for over a year, and 4 had stable disease for over 2 years while on treatment, all despite multiple prior recurrences. Six patients have died, 3 of which had high-grade gliomas at recurrence. Analyses of the first 17 accrued patients showed significant changes in parameters estimated from the DCE data in the T2 lesion in subjects who did not progress in the first 6 months; these changes included reductions in the fractional blood volume (median fBV) and vascular permeability (90th percentile Kps) at 4 months (p Conclusions. In patients with recurrent LGGs treated with everolimus, perfusion parameters may represent early markers of treatment response. Apparent “reversal” of association of p-PRAS40 staining with PFS when everolimus is administered suggests that PI3K/AKT/mTOR pathway activation may serve as a predictive marker of response to mTOR inhibition. Accrual continues and overall efficacy results are pending. Citation Format: Daphne Haas-Kogan, Joanna J. Phillips, Annette Molinaro, Michael Wahl, Sabine Mueller, William A. Weiss, Janine M. Lupo, Mitchel S. Berger, Michael D. Prados, Nicholas Butowski, Jennifer Clarke, Arie Perry, Sarah J. Nelson, Susan Chang. Imaging parameters and molecular correlates as predictive markers of treatment response to mTOR inhibition in adults with recurrent low-grade gliomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2013-3520

Background: Basal cell carcinoma (BCC) is the most common cancer in the United States, with more than two million BCCs diagnosed annually. In recent decades, incidence of BCC has been increasing, particularly among adults under age 40 and especially in young women. Parallel to this incidence trend has been an increase in the popularity of indoor tanning. Although the International Agency for Research on Cancer recently concluded there was “convincing evidence to support a causal association” between indoor tanning and melanoma and squamous cell skin cancer, there was only very limited and inconsistent data for BCC. Methods: We examined indoor tanning in relation to early-onset BCC in the Yale Study of Skin Health in Young People. BCC cases (n=376) and controls with minor benign skin conditions (n=390) under age 40 were identified through Yale Dermatopathology between 2006 and 2010. Approximately two-thirds of dermatologists in Connecticut send their biopsied tissue to Yale dermatopathologists for diagnosis. Controls were frequency matched to cases on age, gender, and body site of biopsy. Participants provided information on ever indoor tanning, age of initiation, frequency, duration, burns while tanning, and type of tanning device during an in-person interview. We calculated odds ratios (OR) and 95% confidence intervals (CI) using multivariate logistic regression with never indoor tanners as the referent group. Results: Ever indoor tanning was associated with a 69% increased risk of early-onset BCC (95% CI = 1.15-2.48); this association was stronger among women (OR = 2.14, 95% CI = 1.31-3.47). The risk from indoor tanning was largely confined to BCCs on the trunk and extremities (OR = 2.81, 95% C I= 1.57-5.02) as opposed to BCCs on the head/neck (OR = 1.11, 95% CI = 0.66-1.86). Indoor tanning was also more strongly associated with multiple BCC (two or more BCCs; 37% of cases) case status (OR = 2.16, 95% CI = 1.26-3.70) than single BCC case status (OR = 1.46, 95% CI = 0.96-2.22). Risk increased dose-dependently with years used regular indoor tanning devices (p-trend = 0.003), number of overall burns (p-trend = Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 635. doi:1538-7445.AM2012-635

Background: In the majority of patients with solid organ malignancies, tumor metastasis marks a transition from curable to terminal. However, a subset of metastatic cancer patients develops only a very limited number of metastases (known as oligometastatic). Survival in this cohort is considerably better than patients with widely disseminated cancer. In fact, in highly select subgroups, as many as 30% of oligometastatic patients are actually cured of cancer. The mechanism that distinguishes limited (or oligometastatic) cancer, from the more lethal, widely disseminated, is unknown. Tumor cell circulation is a critical component of metastasis formation, with circulating tumor cells being detectable in as many as 40-70% of patients with metastatic cancer. We hypothesize that patients with limited metastasis formation, have limited tumor cell circulation. To test this hypothesis, we quantified circulating tumor cells in patients with oligometastatic disease. Methods: Circulating tumor cells (CTCs) were quantified in patients with metastatic epithelial malignancies using the Veridex assay. Patients with > 2 CTCs in a 7.5 ml specimen were considered positive, as a single CTC has previously been shown to carry a substantially higher false positive rate (6.4%) than >2 CTCs (.3%). Results: From April 2008 to December 2009, a total of 20 patients with oligometastatic cancer were enrolled. In all patients the primary tumor had been previously removed and each was being evaluated for curative surgery to remove the oligometastatic cancer. The origin of the primary tumor was colon (17 patients), head and neck (1 patient), ovarian (1 patient), and pancreas (1 patient). The site of oligometastatic cancer was the liver (12 patients), lungs (4 patients) or both liver and lungs (4 patients). Only 1 of the 20 patients with oligometastatic cancer (5%) was found to have >2 circulating tumor cells. During the same time frame, CTCs were more common in patients with disseminated cancer as 4 of 7 patients (57%) with incurable, widely disseminated metastatic cancer had > 2 CTCs (two tailed P value = .0089). Conclusions: Circulating tumor cells are rare in patients with oligometastatic cancer. One mechanism by which oligometastatic cancer patients avoid more widely disseminated cancer may relate to the frequency and duration of tumor cell circulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2234.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces in cultured postmitotic myotubes specific alterations of synthesized proteins as revealed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pulse labeling with [35S]methionine. Synthesis of myosin heavy chain is remarkably inhibited after exposure to 1.6 X 10(-7) M TPA for periods of 9 hr or longer. During shorter periods of TPA treatment (2 hr), an enhanced synthesis of a Mr 31,000 polypeptide is observed, which is associated with the particulate fraction of cultured myotubes. "Pulse chase" experiments show that this polypeptide is not a degradation product induced by TPA. The stimulation of Mr 31,000 polypeptide requires simultaneous RNA synthesis, since actinomycin D completely and selectively abolishes [35S]methionine incorporation into this polypeptide. The stimulation of Mr 31,000 polypeptide is a transient biosynthetic event not detectable after prolonged incubation (24 hr) of myotubes with the tumor promoter. However, TPA-containing medium preincubated with cultures for up to 24 hr induces stimulation of Mr 31,000 polypeptide when administered to untreated cultures. The early stimulatory effect on Mr 31,000 polypeptide synthesis and the late inhibitory effects on contractile protein synthesis are also observed when postmitotic, unfused myoblasts, rather than myotubes, are treated with TPA.